4 The recommended protocol for 1.5-purifications is shown here and described in Section A. Yields are maximized by including the RNA digestion step at the end of the protocol. This enables the RNA to act as a carrier which facilitates DNA precipitation and visualizing the sample pellet. In our experience, downstream applications, like fingerprinting, sequencing and shotgun library construction, are not inhibited by the small amount of residual free nucleotides or the RiboShredder Blend. However, the alternative protocol described in Section B includes an RNA digestion step earlier in the purification process so these residual contaminants are eliminated. 5. General Considerations 1. Optimal cell density: Harvest cells at an OD 600 of 3 to 4 to maximize yields of fosmid DNA. Growing cells for more than 16 hours is not recommended. 2. Avoid shearing: Fosmid DNA, because of its large size, is prone to shearing. Do NOT vortex, shake or pipet the cells after adding FosmidMAX Solution 2 and 3 during the lysis and neutralization steps. The lysis reaction should not exceed 5 minutes. Mix with gentle inversion and use a wide-orifice pipet where noted in the protocol. 3. Proper storage conditions: Store fosmid DNA at 20 C in small aliquots so that repeated freeze-thaw cycles are avoided. 6. Fosmid DNA Purification Protocols A.Purification from 1.5 ml of Culture Note: The protocol given below maximizes yields by including the RNA digestion step at the end of the procedure. This enables the RNA to act as a carrier which facilitates DNA precipitation and visualizing the sample pellet. In our experience, downstream applications, like fingerprinting, sequencing and shotgun library construction, are not inhibited by the small amount of residual free nucleotides or the RiboShredder Blend. However, the alternative protocol described in Section B includes an RNA digestion step earlier in the purification process so these residual contaminants are eliminated. Growing the culture: Prepare 2 ml of LB medium containing the appropriate antibiotic in a 14 ml snap cap culture tube. Inoculate with an isolated colony from a freshly streaked plate and shake (~250 rpm) at 37 C for hours. The culture should be grown to an OD 600 of 3 to 4. Prior to starting: Chill FosmidMAX Solutions 1, 3 and 4 on ice. Dilute RiboShredder RNase Blend 1:4 in TE Buffer. Keep on ice until needed. 1. Transfer 1.5 ml of the overnight culture to a 1.7 ml microcentrifuge tube. Pellet the cells by centrifuging at 15,000 x g or maximum speed for 1-3 minutes. Discard the supernatant. 2. Add 200 μl of chilled FosmidMAX Solution 1 to the pellet. Vortex vigorously to completely resuspend the pellet. Make sure the pellet is completely resuspended before proceeding. 3. Add 400 μl of FosmidMAX Solution 2. Mix by inverting the tube 2-3 times very gently. To avoid shearing the fosmid DNA do NOT vortex, shake or pipet the lysate. Note: The lysis reaction should not exceed 5 minutes. 4

5 4. Add 300 μl of chilled FosmidMAX Solution 3. Mix by inverting the tube 2-3 times very gently. A white precipitate will form in the tube. To avoid shearing the fosmid DNA do NOT vortex, shake or pipet the lysate. 5. Incubate on ice for 15 minutes. 6. Centrifuge at 15,000 x g or maximum speed for 15 minutes at 4 C to pellet cellular debris. 7. Transfer the supernatant to a microcentrifuge tube. Note: To avoid pipetting the white precipitate floating on the surface of the supernatant, place the pipet tip below the meniscus and aspirate slowly, without disturbing the pellet. 8. Add 540 μl or 0.6 volumes of room temperature isopropanol to the recovered supernatant. Mix thoroughly by inverting the tube 4-6 times. 9. Precipitate the nucleic acids by centrifugation at 15,000 x g or maximum speed for 15 minutes at 4 C. Carefully decant the isopropanol. Centrifuge briefly and pipet off any excess isopropanol without disrupting the pellet. 10. Air-dry the pellet at room temperature for 3-5 minutes. Do NOT over-dry or it will be 11. Resuspend the pellet in 250 μl of TE Buffer by tapping and swirling the tube. Make sure the DNA is completely dissolved before proceeding. 12. Add 250 μl of chilled FosmidMAX Solution 4 to the tube. Mix thoroughly by tapping the tube and incubate on ice for 15 minutes. 13. Centrifuge the tube at 15,000 x g or maximum speed for 15 minutes at 4 C. Carefully transfer the supernatant to a microcentrifuge tube without disrupting the pellet. 14. Add 1 ml of absolute ethanol (200 proof) to the recovered supernatant. Mix gently by inverting the tube 4-6 times. 15. Precipitate the DNA by centrifugation at 15,000 x g or maximum speed for 15 minutes at 4 C. Carefully pipette off the ethanol without disrupting the pellet. Centrifuge briefly and pipette off any residual ethanol. 16. Air-dry the pellet at room temperature for 3-5 minutes. Do NOT over-dry or it will be 17. Add 25 μl of TE Buffer to the tube (sterile deionized water or Tris-buffer can also be used) by tapping the tube and leave at room temperature for 10 minutes. 18. Add 1 μl of diluted RiboShredder RNase Blend to the tube and incubate at 37 C for 30 minutes. 19. Quantitate the yield of fosmid DNA by fluorimetry using a DNA specific dye (e.g. PicoGreen or bis-benzimide [Hoechst dye 33258]) or by agarose gel electrophoresis. 20. Store the fosmid DNA at 20 C in small aliquots so that repeated freeze-thaw cycles are avoided. (800)

6 B. Alternative Protocol for Purification from 1.5 ml of Culture Growing the culture: Prepare 2 ml of LB medium containing the appropriate antibiotic in a 14 ml snap cap culture tube. Inoculate with an isolated colony from a freshly streaked plate and shake (~250 rpm) at 37 C for hours. The culture should be grown to an OD 600 of 3 to 4. Prior to starting: Chill FosmidMAX Solutions 1, 3 and 4 on ice. Steps 1-11 are as described in Section A. 12. Add 1 μl of undiluted RiboShredder RNase Blend to the tube and incubate at 37 C for 30 minutes. 13. Add 250 μl of chilled FosmidMAX Solution 4. Mix gently by tapping the tube and incubate on ice for 15 minutes. 14. Centrifuge the tube at 15,000 x g or maximum speed for 15 minutes at 4 C. Carefully transfer the supernatant to a microcentrifuge tube without disrupting the pellet. 15. Add 1 ml of absolute ethanol (200 proof) to the recovered supernatant. Mix gently by inverting the tube 4-6 times. 16. Precipitate the DNA by centrifugation at 15,000 x g or maximum speed for 15 minutes at 4 C. Carefully pipette off the ethanol without disrupting the pellet. Centrifuge briefly and pipette off any residual ethanol. 17. Air-dry the pellet at room temperature for 3-5 minutes. Do NOT over-dry or it will be 18. Resuspend the pellet in 25 μl of TE Buffer (sterile deionized water or Tris-buffer can also be used) by tapping and swirling the tube. 19. Quanitate the yield of fosmid DNA by fluorimetry using a DNA specific dye (e.g. PicoGreen or bis-benzimide [Hoechst dye 33258]) or by agarose gel electrophoresis. 20. Store the fosmid DNA at 20 C in small aliquots so that repeated freeze-thaw cycles are avoided. C. Purification from 40 ml of Culture Growing the culture: Prepare 50 ml of LB medium containing the appropriate antibiotic in a 250 ml flask. Inoculate with an isolated colony from a freshly streaked plate and shake vigorously (~250 rpm) at 37 C for hours. The culture should be grown to an OD 600 of 3 to 4. Prior to starting: Chill FosmidMAX Solutions 1, 3 and 4 on ice. 1. Transfer 40 ml of the overnight culture to a 40 ml Oakridge-style centrifuge tube. Pellet the cells by centrifuging at 5,000 x g for 8 minutes at 4 C. Discard the supernatant. 2. Add 3 ml of chilled FosmidMAX Solution 1 to the pellet. Vortex vigorously to completely resuspend the pellet. Make sure the pellet is completely resuspended before proceeding. 6

7 3. Add 6 ml of FosmidMAX Solution 2. Mix by inverting the tube 2-3 times very gently. To avoid shearing the fosmid DNA do NOT vortex, shake or pipet the lysate. Note: The lysis reaction should not exceed 5 minutes. 4. Add 4.5 ml of chilled FosmidMAX Solution 3. Mix by inverting the tube 2-3 times very gently. A white precipitate will form in the tube. To avoid shearing the fosmid DNA do NOT vortex, shake or pipet the lysate. 5. Incubate on ice for 15 minutes. 6. Centrifuge at 15,000 x g for 15 minutes at 4 C to pellet cellular debris. 7. Transfer the supernatant to a 40 ml Oakridge-style centrifuge tube using a 10 ml pipet. Note: To avoid pipetting the white precipitate floating on the surface of the supernatant, place the pipet tip below the meniscus and aspirate slowly, without disturbing the pellet. 8. Add 8.1 ml or 0.6 volumes of room temperature isopropanol to the recovered supernatant. Mix thoroughly by inverting the tube 4-6 times. 9. Precipitate the nucleic acids by centrifugation at 15,000 x g for 15 minutes at 4 C. Carefully decant the isopropanol. Centrifuge briefly and pipet off any excess isopropanol without disrupting the pellet. 10. Air-dry the pellet at room temperature for 3-5 minutes. Do NOT over-dry or it will be 11. Resuspend the pellet in 500 μl of TE Buffer by tapping and swirling the tube. Make sure the DNA is completely dissolved before proceeding. 12. Add 18 μl of RiboShredder RNase Blend to the tube and incubate at 37 C for 30 minutes. Add an additional 500 μl of TE Buffer and mix by tapping the tube. 13. Add 1 ml of chilled FosmidMAX Solution 4. Mix gently by tapping the tube and incubate on ice for 15 minutes. 14. Centrifuge the tube at 15,000 x g for 15 minutes at 4 C. Carefully transfer the supernatant to a 40 ml Oakridge-style centrifuge tube without disrupting the pellet. 15. Add 4 ml of absolute ethanol (200 proof) to the recovered supernatant in each tube. Mix gently by inverting the tube 4-6 times. 16. Precipitate the DNA by centrifugation at 15,000 x g for 15 minutes at 4 C. Carefully decant the ethanol. Centrifuge briefly and pipette off any residual ethanol without disrupting the pellet. 17. Air-dry the pellet at room temperature for 5-7 minutes. Do NOT over-dry or it will be 18. Add 200 μl of TE Buffer to each tube (sterile deionized water or Tris-buffer can also be used). Resuspend the pellet by tapping the tube and leave the DNA overnight at 4 C to dissolve completely. (800)

8 19. Quantitate the yield of fosmid DNA by fluorimetry using a DNA specific dye (e.g. PicoGreen or bis-benzimide [Hoechst dye 33258]) or by agarose gel electrophoresis. D. Purification from 100 ml of Culture Growing the culture: Prepare 100 ml of LB medium containing the appropriate antibiotic in a 500 ml flask. Inoculate with an isolated colony from a freshly streaked plate and shake vigorously (~250 rpm) at 37 C for hours. The culture should be grown to an OD 600 of 3 to 4. Prior to starting: Chill FosmidMAX Solutions 1, 3 and 4 on ice. 1. Transfer 100 ml of the overnight culture to a 250 ml centrifuge bottle. Pellet the cells by centrifuging at 5,000 x g for 8 minutes at 4 C. Discard the supernatant. 2. Add 6 ml of chilled FosmidMAX Solution 1 to the pellet. Vortex vigorously to completely resuspend the pellet. Make sure the pellet is completely resuspended before proceeding. 3. Transfer equal volumes of the cell suspension (3 ml) to two 40 ml Oakridge-style centrifuge tubes. 4. Add 6 ml of FosmidMAX Solution 2 to each tube. Mix by inverting the tube 2-3 times very gently. To avoid shearing the fosmid DNA do NOT vortex, shake or pipet the lysate. Note: The lysis reaction should not exceed 5 minutes. 5. Add 4.5 ml of chilled FosmidMAX Solution 3 to each tube. Mix by inverting the tube 2-3 times very gently. A white precipitate will form in the tube. To avoid shearing the fosmid DNA do NOT vortex, shake or pipet the lysate. 6. Incubate on ice for 15 minutes. 7. Centrifuge at 15,000 x g for 15 minutes at 4 C to pellet cellular debris. 8. Transfer the supernatant to a 40 ml Oakridge-style centrifuge tube using a 10 ml pipet. Note: To avoid pipetting the white precipitate floating on the surface of the supernatant, place the pipet tip below the meniscus and aspirate slowly, without disturbing the pellet. 9. Add 8.1 ml or 0.6 volumes of room temperature isopropanol to the recovered supernatant. Mix thoroughly by inverting the tube 4-6 times. 10. Precipitate the nucleic acids by centrifugation at 15,000 x g for 15 minutes at 4 C. Carefully decant the isopropanol. Centrifuge briefly and pipet off any excess isopropanol without disrupting the pellet. 11. Air-dry the pellet at room temperature for 3-5 minutes. Do NOT over-dry or it will be 12. Resuspend the pellet in 500 μl of TE Buffer by tapping and swirling the tube. Make sure the DNA is completely dissolved before proceeding. 13. Add 20 μl of RiboShredder RNase Blend to the tube and incubate at 37 C for 30 minutes. Add an additional 500 μl of TE Buffer and mix by tapping the tube. 8

9 14. Add 1 ml of chilled FosmidMAX Solution 4 to each tube. Mix gently by tapping the tube and incubate on ice for 15 minutes. 15. Centrifuge the tube at 15,000 x g for 15 minutes at 4 C. Carefully transfer the supernatant to a 40 ml Oakridge-style centrifuge tube without disrupting the pellet. 16. Add 4 ml of absolute ethanol (200 proof) to the recovered supernatant in each tube. Mix gently by inverting the tube 4-6 times. 17. Precipitate the DNA by centrifugation at 15,000 x g for 15 minutes at 4 C. Carefully decant the ethanol. Centrifuge briefly and pipette off any residual ethanol without disrupting the pellet. 18. Air-dry the pellet at room temperature for 5-7 minutes. Do NOT over-dry or it will be 19. Add 200 μl of TE Buffer to each tube (sterile deionized water or Tris-buffer can also be used). Resuspend the pellet by tapping the tube and leave the DNA overnight at 4 C to dissolve completely. 20. Quantitate the yield of fosmid DNA by fluorimetry using a DNA specific dye (e.g. PicoGreen or bis-benzimide [Hoechst dye 33258]) or by agarose gel electrophoresis. 7. References 1. Epicentre Forum (2002) 9 (1), 3. CopyControl, End-It, EpiFOS, Fast-Link, FosmidMAX, pweb, and RiboShredder are trademarks of Epicentre, Madison, Wisconsin. PicoGreen is a registered trademark of Molecular Probes Inc., Eugene, Oregon. Visit our technical blog: epicentral.blogspot.com (800)

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